APOBEC3G (A3G) and APOBEC3F (A3F), host restriction factor cytidine deaminases,are packaged into HIV-1 virions in the absence of virion infectivity factor (Vif) expression. Vif binds to the APOBEC proteins and induces their degradation, thereby preventing virion incorporation. Our long-term goals are to understand the structure and function of the APOBEC proteins, to elucidate the structural determinants of APOBEC proteins and Vif that interact with each other, and to identify small-molecule inhibitors interfering with this interaction that will form the basis of a new class of antiretroviral drugs. Using mutational analysis, we showed that a single amino acid substitution (D128K) is responsible for the species specificity of A3G proteins. To gain insight into the mechanism by which A3G inhibits HIV-1 replication, we developed a sensitive cytidine deamination assay and demonstrated that interactions with packaged viral and nonviral RNAs are sufficient for A3G virion incorporation, whereas interactions with viral proteins are not essential. To elucidate the mechanism by which A3G inhibits HIV-1 replication in natural target cells of infection, we quantified the amounts of A3G in Vif-deficient virions produced in activated primary CD4+ T cells;these studies indicated that very few molecules (7 4) are packaged into virions during natural infection. In current studies, our extensive mutational analysis of HIV-1 Vif has identified two different determinants that interact with A3F and A3G. We have also used genetic and biochemical approaches to identify domains of A3G and A3F that interact with Vif and characterize these interactions by biochemical and structural approaches. Substitution of amino acids adjacent to D128, most prominently W127, abrogates A3G virion incorporation. We will characterize biochemical and cellular properties of this mutant to understand the mechanism by which A3G is incorporated into virions. To elucidate the mechanism by which A3G inhibits viral replication, we are examining its effects on viral DNA metabolism by quantitative real-time PCR assays. These studies indicate that removal of the primer tRNA during reverse transcription is aberrant and inefficient in the presence of A3G, leading to defects in viral DNA integration and synthesis. A comparison of the mechanism of antiviral inhibition of A3G and A3F has revealed that both proteins inhibit viral DNA integration, but they do so by different mechanisms. Unlike A3G, A3F inhibits provirus formation by inhibiting the 3 processing of viral DNA ends by HIV-1 integrase. We are examining the nucleic acid binding properties of A3G and A3F to understand why these two proteins have different effects on the 3 processing reaction. Using a novel bimolecular fluorescence complementation (BiFC) approach, we have examined the intracellular interactions between A3G proteins, and between A3G and viral proteins and RNA. These studies show that A3G forms multimers in a process that is dependent on RNA binding. We are extending these studies to analyze the interactions between A3G/A3F and Vif proteins, and Vif multimerization. We are also developing in vivo assays for the Vif-A3G and Vif-A3F interactions for high-throughput screening of small molecule inhibitors that block these interactions. We are examining the role of P bodies, recently identified sites of A3G accumulation, in the function of A3G and viral replication. We are purifying A3G to carry out biochemical studies and structural studies using X-ray crystallography. We will determine the affinity of the purified A3G for nucleic acid substrates and other viral proteins such as Gag and Vif. These studies will be used to identify and characterize A3G-Vif interacting domains. [Corresponds to Pathak Project 1 in the April 2007 site visit report of the HIV Drug Resistance Program]APOBEC3G (A3G) and APOBEC3F (A3F), host restriction factor cytidine deaminases,are packaged into HIV-1 virions in the absence of virion infectivity factor (Vif) expression. Vif binds to the APOBEC proteins and induces their degradation, thereby preventing virion incorporation. Our long-term goals are to understand the structure and function of the APOBEC proteins, to elucidate the structural determinants of APOBEC proteins and Vif that interact with each other, and to identify small-molecule inhibitors interfering with this interaction that will form the basis of a new class of antiretroviral drugs. Using mutational analysis, we showed that a single amino acid substitution (D128K) is responsible for the species specificity of A3G proteins. To gain insight into the mechanism by which A3G inhibits HIV-1 replication, we developed a sensitive cytidine deamination assay and demonstrated that interactions with packaged viral and nonviral RNAs are sufficient for A3G virion incorporation, whereas interactions with viral proteins are not essential. To elucidate the mechanism by which A3G inhibits HIV-1 replication in natural target cells of infection, we quantified the amounts of A3G in Vif-deficient virions produced in activated primary CD4+ T cells;these studies indicated that very few molecules (7 4) are packaged into virions during natural infection. In current studies, our extensive mutational analysis of HIV-1 Vif has identified two different determinants that interact with A3F and A3G. We have also used genetic and biochemical approaches to identify domains of A3G and A3F that interact with Vif and characterize these interactions by biochemical and structural approaches. Substitution of amino acids adjacent to D128, most prominently W127, abrogates A3G virion incorporation. We will characterize biochemical and cellular properties of this mutant to understand the mechanism by which A3G is incorporated into virions. To elucidate the mechanism by which A3G inhibits viral replication, we are examining its effects on viral DNA metabolism by quantitative real-time PCR assays. These studies indicate that removal of the primer tRNA during reverse transcription is aberrant and inefficient in the presence of A3G, leading to defects in viral DNA integration and synthesis. A comparison of the mechanism of antiviral inhibition of A3G and A3F has revealed that both proteins inhibit viral DNA integration, but they do so by different mechanisms. Unlike A3G, A3F inhibits provirus formation by inhibiting the 3 processing of viral DNA ends by HIV-1 integrase. We are examining the nucleic acid binding properties of A3G and A3F to understand why these two proteins have different effects on the 3 processing reaction. Using a novel bimolecular fluorescence complementation (BiFC) approach, we have examined the intracellular interactions between A3G proteins, and between A3G and viral proteins and RNA. These studies show that A3G forms multimers in a process that is dependent on RNA binding. We are extending these studies to analyze the interactions between A3G/A3F and Vif proteins, and Vif multimerization. We are also developing in vivo assays for the Vif-A3G and Vif-A3F interactions for high-throughput screening of small molecule inhibitors that block these interactions. We are examining the role of P bodies, recently identified sites of A3G accumulation, in the function of A3G and viral replication. We are purifying A3G to carry out biochemical studies and structural studies using X-ray crystallography. We will determine the affinity of the purified A3G for nucleic acid substrates and other viral proteins such as Gag and Vif. These stu [summary truncated at 7800 characters]